This invention relates to a clotting assay performed using freshly obtained, non-anticoagulated, blood and more particularly to a whole blood Activated Partial Thromboplastin Time (APPT) test which is performed using fresh non-citrated whole blood and specific reagents placed in a single test tube.
Fundamental to human lift and well being is the ability of human blood, in response to certain stimuli, to thicken and eventually form structures known as blood clots. Blood clotting occurs in response to both internal and external bleeding. Conversely, unwanted clot formation or blood thickening can have undesirable effects among which are circulatory blockages. Clinical occurrence of uncontrolled bleeding or clotting are corrected by therapeutic infusion of blood, blood products or medications. All therapies are monitored to determine efficacy by use of specific blood clotting assays.
Blood clotting is a process which involves three interacting components. These are the blood vessels, blood coagulation factors and blood platelets. The blood coagulation factors are proteins or glycoproteins which freely circulate within the body. In the clot formation process they interact in a mechanism commonly referred to as the coagulation cascade. In this process an inactive coagulation factor is chemically converted to an active enzyme. This enzyme subsequently converts another inactive enzyme precursor to an active state. The end result of these processes is the conversion of plasma soluble fibrinogen to fibrin, which is plasma insoluble. The fibrin clot is a crosslinked matrix which entraps the formed elements of the blood thereby sealing off the bleeding site. The formed elements consist of the platelets, white blood cells and red blood cells.
Platelets are cell fragments which play multiple roles in the clotting process. They initially attach to the exposed collagen matrix of broken blood vessels and to each other to form a primary platelet plug to seal off the bleeding site. During this aggregation the platelets release chemical components into the plasma which are important in the clotting process. One such component is called platelet factor 3 (PF3). Platelet factor 3 is a phospholipid which serves as a necessary cofactor in the coagulation cascade.
Specific blood clotting assays enable the clinician to determine the integrity of the blood coagulation cascade and the efficacy of therapy. One such clotting assay is the Partial Thromboplastin Time (PTT) test. This test was first described by Brinkhous and Langdell. See an article entitled "Newer Approaches To The Study Of Hemophilia & Hemophiloidal States" by K. M. Brinkhous et al, JAMA 154:481-486, 1954. See an article entitled "Effect of Antihemophilic Factor On One Stage Clotting Tests" by R. D. Langdell et al, J Lab Clin Med 41:637-647, 1953. The principle of the PTT is that citrated platelet poor (i.e., depleted) plasma (PPP) is added to a mixture containing platelet factor 3 (PF3) substitute and calcium. The PF3 substitute is a phospholipid derived from brain and lung tissue. See article entitled "A Brain Extract As A Substitute For Platelet Suspensions In The Thromboplastin Generation Test" by W. H. Bell and H. G. Alton, Nature 174:880-881, 1954. This substitutes for the role which platelets play in the clotting process. The calcium is required since the native calcium present in circulating blood has been rendered unusable by the addition of citrate. Calcium is a necessary component for clotting.
The PTT assay proved a valuable clinical test to evaluate the coagulation cascade. Its one major drawback was a relative lack of reproducibility among different individuals. This made it difficult to establish a range of normal values of the test and determine if a patient's value was within or outside of this normal range. With further research it was determined that much of this variability was attributable to the processes involved in the initial activation of the coagulation cascade. This "contact activation" sequence was found to be highly variable among individuals. By standardizing the rate at which this activation occurs, it was possible to greatly improve the reliability of the PTT assay. This standardization was accomplished by addition of an activator to the PTT mixture. The PTT conducted in the presence of this activator was first described by Proctor and Rapaport. See an article entitled "The Partial Thromboplastin Time With Kaolin" by R. P. Proctor et al, Am J Clin Path 36:212-219, 1961. This test is known as the Activated Partial Thromboplastin Time (APTT) test. The test employed the particulate activator kaolin and later studies demonstrated the use of a soluble plasma activator, ellagic acid. See an article entitled "Activation of Hageman Factor by Solutions of Ellagic Acid" by O. D. Ratnoff and J. D. Crum, J Lab Clin Med 63:359-377, 1964. The APTT assays currently used in the clinical laboratory are minor modifications of these earliest tests. See the chapter, "Recalcification Time Test And Its Modification (Partial Thromboplastin Time, Activated Partial Thromboplastin Time And Expanded Partial Thromboplastin Time", by C. Hougie, In the text Hematology, 3rd edition, ed Williams, McGraw Hill Book Co., N.Y., pg. 1662-1664, (1983).
By elimination of the variable nature of activation, the APTT test has proven a more reliable test than the earlier PTT. It is particularly useful in identification of clotting factor deficiencies, of which the most common are the Hemophilias--Hemophilia A (Factor VIII deficiency), Hemophilia B (Factor IX deficiency) and Hemophilia C (Factor XI deficiency). It is also valuable as means to monitor the effect of clot inhibiting agents such as heparin. Heparin is an animal derived substance which directly interferes with the formation of the fibrin clot and is used extensively to control clotting in patients predisposed to uncontrolled coagulation. Such patients are typically those having a recent myocardial infarction (i.e., heart attack), stroke or episode of thrombophlebitis (clots in the large blood vessels). Another group of patients that require heparin are those individuals who require extracorporeal circulation, i.e., heart bypass surgery or renal dialysis.
To date the APTT assay has been performed exclusively using citrated platelet poor plasma. Such blood is routinely obtained by hospital personnel and transported to a central laboratory where the test is performed. Such batch processing has proven economical and practical to screen large numbers of patient plasmas. However the same characteristics of the test which make it practical in this example render it unacceptable in a variety of clinical settings. For example in open heart surgery the delay of time between drawing the blood sample and obtaining the APTT result makes the test useless in control of therapy. To overcome these drawbacks a more practical test used in this setting is the Activated Clotting Time Test (ACT) (Hattersley, JAMA 196:150-154, 1966). Unlike the APTT this test is performed at the patient's bedside using native, or, non-anticoagulated (non-citrated) blood. Such an assay allows one to quickly assess the effect of heparin on the clotting mechanism and to alter the therapy appropriately. This test also allows one to measure the high levels of heparin used in these instances.
An obvious difference between the ACT and APTT is that clot formation proceeds in the ACT in the presence of plasma plus all blood elements. Scientists have advocated that such an analysis is more indicative of the patient's true coagulation state than clot inducing formation in plasma. Consequently, the APTT using whole blood has been proposed as a more sensitive and accurate measure of the coagulant state than the plasma based ACT (P. Hattersley, Heparin Anticoagulation, in the text Laboratory Hematology, ed. by J. Koepke, Churchill, Livingston, p. 789-818, 1984). In one such protocol the use of which has not been well documented, whole blood samples are collected from the patient and citrate anticoagulated. The blood is then transferred to a reaction vessel to which the APTT reagents and calcium is subsequently added. This two step process is necessary as a meaningful APTT test requires a period of contact activation which traditionally ranges from 2 to 5 minutes. In order to achieve uniformity of activation, the clotting process is inhibited by sequestering of calcium with citrate or unregulated coagulation will occur. The two step whole blood protocol is therefore essentially identical to the plasma APTT. In a second version non-anticoagulated blood is obtained from the patient to perform a bedside test. (J. Blakely, A Rapid Bedside Method For Control Of Heparin Therapy, Canad. Med. Assoc. J. 99:1072-1076, 1968). The non-citrated blood is mixed with activator and PF.sub.3 substrate and the clotting time determined. The lack of citrate anticoagulation eliminates the ability to regulate the "contact activation" and this test, therefore, bears more resemblance to the ACT than the APTT.
The present invention relates to an APTT assay which does not require blood which has previously been anticoagulated with citrate. By combining the citrate anticoagulation step with the contact activation step an APTT assay is provided which uses fresh, non-anticoagulated blood specimens. Thus, the coagulant state in whole blood may be defined independently of the previous need to pre-collect blood in a special citrated tube.
The major distinguishing feature of the APTT test compared to its forefather, the PTT test, is that a protocol is followed which allows the blood to be in contact for a pre-established, standardized period of time with a blood coagulation activator. This period of "contact activation" is a time and temperature dependent process during which the coagulation cascade is initialized. In this manner, the time to formation of a clot, following the addition of calcium, is truly indicative of the integrity of the clotting state.
Since blood removed from the body has a natural tendency to clot, it is imperative that a substance be added to prevent uncontrolled coagulation. In order to properly perform the APTT test, blood must be anticoagulated so that the contact activation step may be properly performed.
Many substances have the inherent ability to prevent clotting such as heparin, dextran, EDTA or sodium oxylate. However the anticoagulant of choice is sodium citrate. This substance works exclusively by chelating the free calcium ions present in the blood. Without calcium, clotting cannot take place. The other anticoagulants are not used since they exert multiple effects which disrupt normal coagulation processes. The anticoagulant effect of citrate is easily reversed by addition of exogenous calcium. Calcium chloride is the salt commonly used. The present invention contains the step of citrate anticoagulation within a test container whereby an APTT assay may be performed at the patient bedside on non-anticoagulated whole blood specimens which are drawn directly from the patient. Consequently, the advantages of an immediate, bedside analysis of patient samples, which the ACT brought to open heart surgery are now available to the hospitalized community at large and to out patients in clinics and doctor's offices.
Prior to the development of the assay according to this invention, the APTT has been traditionally performed using citrated plasma. Though occasionally advocated for use with citrated whole blood, the test is rarely performed on this substrate and has not been clinically documented.